The measurement of testosterone (T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women. Circulating T is bound with high affinity to sex hormone binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active. Conditions that affect SHBG concentrations, such as aging and obesity, alter total but not free T concentrations; in these conditions, the determination of free T is necessary to obtain an accurate assessment of androgen status. Tracer analog method, the most widely used method for free T, has been shown to be inaccurate. Equilibrium dialysis method, considered the reference method, is technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that ??the calculation of free testosterone is the most useful estimate of free testosterone in plasma?? Therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis. We have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterone's binding to SHBG using modern biophysical techniques. We have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer. Our computational frame-work incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T:SHBG association, and allsotery. In phase I studies, we demonstrated that TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogoandal men. We have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect, and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis. The phase I studies have led to adoption of the TruTTM algorithm at several institutions. The phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentrations, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (Aim 2). We will generate population-based reference ranges for free T (Aim 3). Phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption. The phase II program will provide validation of TruTTM algorithm in the two most common clinical indications for free T measurement ? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders. It will also enable the development of a HIPAA compliant platform that can be embedded into electronic medical record for wider clinical adoption and for improving clinical care.